Jürg Streit

The generation of rhythmic activity in dissociated cultures of rat spinal cord

Locomotion in vertebrates is controlled by central pattern generators in the spinal cord. The roles of specific network architecture and neuronal properties in rhythm generation by such spinal networks are not fully understood. We have used multisite recording from dissociated cultures of embryonic rat spinal cord grown on multielectrode arrays to investigate the patterns of spontaneous activity in randomised spinal networks. We were able to induce similar patterns of rhythmic activity in dissociated cultures as in slice cultures, although not with the same reliability and not always with the same protocols. The most reliable rhythmic activity was induced when a partial disinhibition of the network was combined with an increase in neuronal excitability, suggesting that both recurrent synaptic excitation and neuronal excitability contribute to rhythmogenesis. During rhythmic activity, bursts started at several sites and propagated in variable ways. However, the predominant propagation patterns were independent of the protocol used to induce rhythmic activity. When synaptic transmission was blocked by CNQX, APV, strychnine and bicuculline, asynchronous low‐rate activity persisted at ≈ 50% of the electrodes and ≈ 70% of the sites of burst initiation. Following the bursts, the activity in the interval was transiently suppressed below the level of intrinsic activity. The degree of suppression was proportional to the amount of activity in the preceding burst. From these findings we conclude that rhythmic activity in spinal cultures is controlled by the interplay of intrinsic neuronal activity and recurrent excitation in neuronal networks without the need for a specific architecture.

Spatiotemporal characterization of rhythmic activity in rat spinal cord slice cultures

Rat spinal networks generate a spontaneous rhythmic output directed to motoneurons under conditions of increased excitation or of disinhibition. It is not known whether these differently induced rhythms are produced by a common rhythm generator. To investigate the generation and the propagation of rhythmic activity in spinal networks, recordings need to be made from many neurons simultaneously. Therefore extracellular multisite recording was performed in slice cultures of embryonic rat spinal cords grown on multielectrode arrays. In these organotypic cultures most of the spontaneous neural activity was nearly synchronized. Waves of activity spread from a source to most of the network within 35–85 ms and died out after a further 30–400 ms. Such activity waves induced the contraction of cocultured muscle fibres. Several activity waves could be grouped into aperiodic bursts. Disinhibition with bicuculline and strychnine or increased excitability with high K+ or low Mg2+ solutions could induce periodic bursting with bursts consisting of one or several activity waves. Whilst the duration and period of activity waves were similar for all protocols, the duration and period of bursts were longer during disinhibition than during increased excitation. The sources of bursting activity were mainly situated ventrally on both sides of the central fissure. The pathways of network recruitment from one source were variable between bursts, but they showed on average no systematic differences between the protocols. These spatiotemporal similarities under conditions of increased excitation and of disinhibition suggest a common spinal network for both types of rhythmic activity.

Mechanisms controlling bursting activity induced by disinhibition in spinal cord networks

Disinhibition reliably induces regular synchronous bursting in networks of spinal interneurons in culture as well as in the intact spinal cord. We have combined extracellular multisite recording using multielectrode arrays with whole cell recordings to investigate the mechanisms involved in bursting in organotypic and dissociated cultures from the spinal cords of embryonic rats. Network bursts induced depolarization and spikes in single neurons, which were mediated by recurrent excitation through glutamatergic synaptic transmission. When such transmission was blocked, bursting ceased. However, tonic spiking persisted in some of the neurons. In such neurons intrinsic spiking was suppressed following the bursts and reappeared in the intervals after several seconds. The suppression of intrinsic spiking could be reproduced when, in the absence of fast synaptic transmission, bursts were mimicked by the injection of current pulses. Intrinsic spiking was also suppressed by a slight hyperpolarization. An afterhyperpolarization following the bursts was found in roughly half of the neurons. These afterhyperpolarizations were combined with a decrease in excitability. No evidence for the involvement of synaptic depletion or receptor desensitization in bursting was found, because neither the rate nor the size of spontaneous excitatory postsynaptic currents were decreased following the bursts. Extracellular stimuli paced bursts at low frequencies, but failed to induce bursts when applied too soon after the last burst. Altogether these results suggest that bursting in spinal cultures is mainly based on intrinsic spiking in some neurons, recurrent excitation of the network and auto‐regulation of neuronal excitability.

Voltage dependent calcium currents in PC12 growth cones and cells during NGF-induced cell growth

The role of calcium currents in the regulation of neurite outgrowth is still rather speculative. As a contribution to this field, macroscopic voltage dependent calcium currents were investigated in relation to the nerve growth factor (NGF)-induced outgrowth of neurites in PC 12 cells. Calcium currents were recorded in isolated growth cones of PC 12 cells using the whole cell patch clamp method. The currents were activated at high voltages and only slightly inactivated with time. The currents were identical to those found in the cell soma of PC 12 cells and similar to the classical high-voltage-activated calcium current found in many neuronal cells. The peak current density in the growth cones was in the same range as in the cell somata. The calcium currents of the cell somata were not modified during the early phase of NGF application, despite the occurrence of NGF-induced soma growth and outgrowth of neurites. The current density at this time was therefore lower in NGF-treated cells than in untreated cells. In a later phase, maximal current amplitudes of NGF-treated cells were higher than in untreated cells indicating an increase in current density to values similar to that found in the untreated cells. In addition, the calcium current inactivation was found to be more pronounced in the NGF-treated cells by that time. The results are discussed with regard to a possible role of calcium currents in the regulation of NGF-induced neurite outgrowth in these cells.

INaP underlies intrinsic spiking and rhythm generation in networks of cultured rat spinal cord neurons

We have shown previously that rhythm generation in disinhibited spinal networks is based on intrinsic spiking, network recruitment and a network refractory period following the bursts. This refractory period is based mainly on electrogenic Na/K pump activity. In the present work, we have investigated the role of the persistent sodium current (INaP) in the generation of bursting using patch‐clamp and multielectrode array recordings. We detected INaP exclusively in the intrinsic spiking cells. The blockade of INaP by riluzole suppressed the bursting by silencing the intrinsic spiking cells and suppressing network recruitment. The blockade of the persistent sodium current produced a hyperpolarization of the membrane potential of the intrinsic spiking cells, but had no effect on non‐spiking cells. We also investigated the involvement of the hyperpolarization‐activated cationic current (Ih) in the rhythmic activity. The bath application of ZD7288, a specific Ih antagonist, slowed down the rate of the bursts by increasing the interburst intervals. Ih was present in ∼ 70% of the cells, both in the intrinsic spiking cells as well as in the non‐spiking cells. We also found both kinds of cells in which Ih was not detected. In summary, in disinhibited spinal cord cultures, a persistent sodium current underlies intrinsic spiking, which, via recurrent excitation, generates the bursting activity. The hyperpolarization‐activated cationic current contributes to intrinsic spiking and modulates the burst frequency.

Transmitter concentration profiles in the synaptic cleft: an analytical model of release and diffusion

A three-dimensional model for release and diffusion of glutamate in the synaptic cleft was developed and solved analytically. The model consists of a source function describing transmitter release from the vesicle and a diffusion function describing the spread of transmitter in the cleft. Concentration profiles of transmitter at the postsynaptic side were calculated for different transmitter concentrations in a vesicle, release scenarios, and diffusion coefficients. From the concentration profiles the receptor occupancy could be determined using alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor kinetics. It turned out that saturation of receptors and sufficiently fast currents could only be obtained if the diffusion coefficient was one order of magnitude lower than generally assumed, and if the postsynaptic receptors formed clusters with a diameter of roughly 100 nm directly opposite the release sites. Under these circumstances the gradient of the transmitter concentration at the postsynaptic membrane outside the receptor clusters was steep, with minimal cross-talk among neighboring receptor clusters. These findings suggest that for each release site a corresponding receptor aggregate exists, subdividing an individual synapse into independent functional subunits without the need for specific lateral diffusion barriers.

A modified roller tube technique for organotypic cocultures of embryonic rat spinal cord, sensory ganglia and skeletal muscle

The roller tube technique as initially described in the literature in 1981, was modified in several aspects for the coexplantation of embryonic rat spinal cord with attached dorsal root ganglia and skeletal muscle from newborn rats. The high metabolic activity of this coculture system required a particular culturing protocol to stabilize pH and osmotic pressure. The appropriate adjustment of the partial pressure of carbon dioxide gas in the incubator proved to be essential for the control of the pH within narrow limits (7.3 +/- 0.1). The adjustment of the osmotic pressure of the medium (290-300 mOsm) improved the growth of the cultures considerably. Roller drum speed was set to 120 revolutions per hour for enhanced flattening of the culture. A simple rating system was used to evaluate neuronal and non-neuronal outgrowth under different modifications of the culture system. Furthermore, morphological and electrophysiological criteria were defined for evaluating individual neurons. The technique described insures the growth of long-term organotypic cocultures of spinal cord, sensory ganglia and skeletal muscle.

An Organotypic Spinal Cord‐Dorsal Root Ganglion‐Skeletal Muscle Coculture of Embryonic Rat. I. The Morphological Correlates of the Spinal Reflex Arc

The cytoarchitecture of a spinal cord‐dorsal root ganglion ‐ skeletal muscle tissue coculture system was investigated at the level of the light microscope using a number of different staining techniques. In these cultures central synapses between dorsal root ganglion (DRG) cells and interneurons in the ventral spinal cord and between DRG cells and motoneurons were visualized by parvalbumin immunostaining and by intracellular horseradish peroxidase (HRP) filling of DRG cells. Skeletal muscle fibres regenerated in vitro first into multinucleated myotubes, and around day 8 in vitro into well differentiated muscle fibres with regular cross‐striation. At the same time newly formed motor endplates could be visualized using acetylcholinesterase staining. The axons of motoneurons could be traced retrogradely by local application of HRP to the regenerated muscle fibres. The motor axons sometimes gave off collaterals reminiscent of Renshaw collaterals at about 300 μm from the axon hillock. Intracellular filling of motoneurons with HRP revealed that only a minority of the motoneurons within a culture had reached their appropriate target. Comparing the dendrograms of the motoneurons which had innervated muscles to those which had not suggested that motoneurons innervating muscle tissue had more complex dendritic trees and larger somata than those which did not innervate muscle tissue. Peripheral neurites of parvalbumin‐immunoreactive DRG cells coiling around regenerated muscle fibres could be demonstrated in these cultures. These probably correspond to that part of the sensory muscle spindle apparatus which developed in vivo. However, only a few of the several hundred DRG cells found in every culture were parvalbumin‐immunoreactive, suggesting that the actual number of la and II afferents within the population of DRG cells in culture is very small. This study demonstrates that all the neural elements necessary for the segmental spinal reflexes develop and can be maintained for several weeks in vitro.

An Organotypic Spinal Cord‐Dorsal Root Ganglion‐Skeletal Muscle Coculture of Embryonic Rat. II. Functional Evidence for the Formation of Spinal Reflex Arcs In Vitro

Electrical properties of motoneurons, muscle fibres and dorsal root ganglion (DRG) cells were studied in an organotypic coculture of embryonic rat spinal cord, dorsal root ganglia and skeletal muscle. The motoneurons were identified by their morphology and position in culture. Their size and input conductance were significantly larger than those of spinal interneurons. Intracellular current injection evoked action potentials in all motoneurons, but only evoked stable repetitive firing patterns in some. Excitability was correlated to somatic size and the rate of spontaneous excitatory input. It is suggested that the somatic growth and the increase in excitability is regulated by the excitatory afferents. The motoneurons showed spontaneous excitatory and inhibitory postsynaptic potentials and action potentials which disappeared with the application of various agents known to inhibit excitability or excitatory synaptic transmission. Excitatory and inhibitory postsynaptic potentials (EPSPs and IPSPs respectively) were distinguished by their shape, reversal potential and pharmacology. IPSPs could be depolarizing or hyperpolarizing in different cells. A higher percentage of cells with hyperpolarizing IPSPs was found in older cultures and in the presence of skeletal muscle, suggesting a reversal of the polarity of IPSPs with development. The spontaneous muscle contractions observed in the cultures could be due either to innervation, spontaneous oscillations of the membrane potential, or electrical coupling between neighbouring fibres. A small percentage of DRG cells showed spontaneous action potentials, all of which were found in cultures with spontaneous muscle contractions. The electrical stimulation of DRG afferents evoked mono‐ and polysynaptic EPSPs in motoneurons, endplate potentials and muscle contractions. The stimulation of the ventral horns evoked endplate potentials and muscle contractions via mono‐ or polysynaptic pathways. Together these results indicate that appropriate and functional contacts were established in the culture between myotubes and DRG cells, between DRG cells and motoneurons, and between motoneurons and muscle fibres.

Dynamics of a random neural network with synaptic depression

We consider a randomly connected neural network with linear threshold elements which update in discrete time steps. The two main features of the network are: (1) equally distributed and purely excitatory connections and (2) synaptic depression after repetitive firing. We focus on the time evolution of the expected network activity. The four types of qualitative behavior are investigated: singular excitation, convergence to a constant activity, oscillation, and chaos. Their occurrence is discussed as a function of the average number of connections and the synaptic depression time. Our model relies on experiments with a slice culture of disinhibited embryonic rat spinal cord. The dynamics of these networks essentially depends on the following characteristics: the low non-structured connectivity, the high synaptic depression time and the large EPSP with respect to the threshold value.